PetChek HTWM PF

The PetChek Canine Heartworm Antigen Test is an enzyme immunoassay for the detection of Dirofilaria immitis (D. immitis) antigen in canine or feline serum or plasma. A microtitration format has been devised in which antibodies to D. immitis antigen are coated on test wells. Upon incubation of the sample in the coated test well, antigen in the sample forms complexes with the coated antibodies.

Following removal of the sample, an enzyme conjugated antibody is added and binds to antigen captured in the well. In the final step of the assay, unbound conjugate is washed away and enzyme substrate/chromogen is added. Subsequent color development indicates the presence of heartworm antigen in the sample.

Precautions And Warnings

- Do not expose TMB Substrate Solution to direct sunlight or any oxidizing agents.

- Care should be taken to prevent contamination of kit components.

- Optimal results will be obtained by strict adherence to this procedure. Careful pipetting and washing technique throughout this procedure are necessary to maintain precision and accuracy.

Storage

Store all reagents at 2-8°C. Allow reagents to come to room temperature (18-25°C) before use. Precipitation may occur in Stop Solution. Bring to room temperature (18-25°C) and mix by swirling before use.

Materials Required But Not Provided

Use Of Precision Pipette To Dispense 100 µl

Place disposable pipette tip on pipette. Depress button on pipette, place tip in sample and release button slowly to pull up sample. Depress button to dispense sample in well.

Preparation Of Wash Solution

Dilute Wash Concentrate ten-fold (1/10) with distilled/deionized water (e.g. 5 mL of Wash Concentrate plus 45 mL of water for each 12 wells). Salt crystals may form in the Wash Concentrate upon storage. If this occurs, allow concentrate to come to room temperature (18-25°C) and mix by swirling to redissolve crystals before preparing wash solution.

Sample Information

Serum or plasma may be used in this test. Serum or plasma (e.g., Heparin, EDTA) may be stored for up to 7 days at 2-8°C. Highly hemolyzed samples should not be used, however moderately hemolyzed or lipemic samples will not affect results. For longer storage, sample should be frozen (-20°C or colder).

Test Procedure

1. Count the number of samples to be tested plus two wells for the positive and negative controls (one each). If using the well holder, remove the required number of wells from the bag and place into the well holder (up to 2 strips or 24 wells). If using the plate frame, remove the required number of complete strips from the bag and place into the 96-well plate frame. Record the positions of samples and controls on a worksheet.

2. Add 2 drops (100 µL) of Positive Control to the first well and 2 drops (100 µL) of Negative Control to the second well.

3. Using the precision pipette provided in kit and a separate pipette tip for each sample, add 100 µL of serum or plasma to appropriate wells. Incubate sample in well for 5 minutes at room temperature (18-25°C).

4. Discard the fluid from the wells by inverting. Slap firmly onto absorbent paper to remove all fluid.

6. Discard the fluid from the wells by inverting. Slap firmly onto absorbent paper to remove all fluid. Wash wells with a forceful stream of diluted wash solution by completely filling each well and discarding the fluid. Slap wells onto absorbent paper after each wash. Repeat for a total of 5 wash cycles.

8. Add 1 drop (50 µL) of Stop Solution to each well. Mix by tapping gently. Read result visually. Color will be stable for 15 minutes.

Results

A sample is considered positive if it has more color than the negative control. For the assay to be valid, the positive control must develop a distinct blue color. Negative control must be clear or very lightly colored.

All positive samples that have not been run in duplicate should be retested. If duplicate samples or repeat testing yield inconsistent results, repeat test.

Sensitivity and Specificity

Sensitivity and Specificity of Heartworm PetChek

Using Characterized Positive and Negative Samples

Sample Size
Kit/Reference

Sample Type

Relative Sensitivity and Specificity
95% Confidence Limit

Kappa Statistic

+/+

-/+

+/-

-/-

Study ID1

59

1

0

65

Serum

Sen. 98% (95% CL 91.1-100%)
Spec. 100% (95% 99.5-100%)

0.984

1. Based on a comparison of four in-clinic heartworm tests, in which any samples yielding discrepant results were further characterized as positive or negative by necropsy.

CL = Confidence limit

Symbol Descriptions

IDEXX Customer Support

USA/Canada 1 800 248 2483

Europe 00800 1234 3399

Australia 1300 44 33 99

idexx.com

U.S. Vet. License No. 313

Product Code 5018.02

*PetChek is a trademark or registered trademark of IDEXX Laboratories, Inc. or its affiliates in the United States and/or other countries.

Every effort has been made to ensure the accuracy of the PetChek HTWM PF information published above. However, it remains the responsibility of the readers to familiarize themselves with the product information contained on the US product label or package insert.

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